Purpose

To keep and increase spermatogonial stem cell number (SSC) is the only available option for pediatric cancer survivors to maintain fertility. Leptin is secreted by the epididymal white adipose tissue and has receptors on stem/progenitor spermatogonia. The purpose of this study is to demonstrate dose- and time-dependent proliferative effect of leptin on stem/progenitor spermatogonia cultures from prepubertal mice testes.

Methods

CD90.2 (+) stem/progenitor spermatogonia were isolated from the C57BL/6 mouse testis on postnatal day 6 and placed in culture. The proliferative effect of leptin supplementation was assessed by colony formation (diameter and number), WST proliferation assays, and xCELLigence real-time cell analysis (RTCA) on days 3, 5, and 7 of culture. Expressions of p-ERK1/2, p-STAT3, total STAT3, and p-SHP2 levels were determined by western blot analysis.

Results

Leptin supplementation of 100 ng/ml increased the diameter (p = 0.001) and number (p = 0.01) of colonies in stem/progenitor spermatogonial cultures and caused higher proliferation by WST-1 (p = 0.009) compared with the control on day 7. The EC50 was calculated as 114 ng/ml for leptin by RTCA. Proliferative dose of leptin induced increased expression of p-ERK1/2 (p = 0.009) and p-STAT3 (p = 0.023) on stem/progenitor spermatogonia when compared with the untreated group.

Conclusion

The results indicated that leptin supplementation exhibited a dose- and time-dependent proliferative effect on stem/progenitor spermatogonia that was associated with increased expression of ERK1/2 and STAT3 pathways while maintaining their undifferentiated state. This output presents a new agent that may help to expand the stem/progenitor spermatogonia pool from the neonatal testis in order to autotransplant after cancer treatment.

中文翻译：

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瘦素促进新生小鼠干细胞/祖细胞精原细胞的增殖。

目的

保持和增加精原干细胞数量 (SSC) 是儿童癌症幸存者保持生育能力的唯一选择。瘦素由附睾白色脂肪组织分泌，在干/祖精原细胞上有受体。本研究的目的是证明瘦素对青春期前小鼠睾丸干/祖精原细胞培养物的剂量和时间依赖性增殖作用。

方法

CD90.2 (+) 茎/祖精原细胞在出生后第 6 天从 C57BL/6 小鼠睾丸中分离出来并置于培养物中。在培养的第 3、5 和 7 天，通过集落形成（直径和数量）、WST 增殖测定和 xCELLigence 实时细胞分析 (RTCA) 评估补充瘦素的增殖效果。通过蛋白质印迹分析确定 p-ERK1/2、p-STAT3、总 STAT3 和 p-SHP2 水平的表达。

结果

与第 7 天的对照相比，补充 100 ng/ml 的瘦素增加了茎/祖细胞精原细胞培养物中集落的直径 ( p = 0.001) 和数量 ( p = 0.01)，并通过 WST-1 引起更高的增殖 ( p = 0.009) . 通过 RTCA 计算瘦素的 EC50 为 114 ng/ml。与未治疗组相比，瘦素的增殖剂量诱导干/祖精原细胞上 p-ERK1/2 ( p = 0.009) 和 p-STAT3 ( p = 0.023)的表达增加。

结论

结果表明，补充瘦素对干/祖细胞精原细胞具有剂量和时间依赖性增殖作用，这与 ERK1/2 和 STAT3 通路的表达增加有关，同时保持其未分化状态。此输出提供了一种新试剂，可能有助于扩大新生儿睾丸的干/祖精原细胞池，以便在癌症治疗后进行自体移植。